This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A previous EXAFS investigation reported that the thiolate-ligated Fe(IV)oxo (ferryl) species in chloroperoxidase compound II is protonated at pH 6.5. The importance of this observation lies in its connection to C-H bond activation. The ability of metal-oxos to abstract hydrogen scales with the strength of the O-H bond formed;in oxidative heme enzymes, this depends on the reduction potential of compound I (a ferryl-radical species) and the pKa of compound II (a ferryl species). Subsequent Mossbauer measurements of chloroperoxidase compound II have revealed the presence of two distinct ferryl species in an . 70:30 ratio. We have found the 70:30 ratio of component concentrations to be independent of pH, and we have obtained the same 70:30 ratio with different oxidants (peroxide and peracetic acid) and reductants (ascorbate and p-phenolsulfonic acid). Recently, we have determined a means of preparing a single component of CPO-II. Reaction of CPO with meta-chloroperbenzoic acid and ascorbate results in a single ferryl species. The Mossbauer parameters of this intermediate are identical to those of the major component of CPO-II. We have also found that we can selectively access the major component of CPO-II through the chemical modification of chloroperoxidase. The reaction of CPO with diethylpyrocarbonate modifies histidine residues. One of these His residues is in a proton-shuttle near the CPO activesite. The reaction of modified CPO with peracetic acid and ascorbate yields a species (. 90% yield) with Mossbauer parameters that are virtually identical to the parameters measured for the m-CPBA intermediate and the major component of CPO-II. We propose an EXAFS examination of the intermediates prepared by 1) reaction of m-CPBA with CPO and 2) chemical modification of the proton shuttle. We seek to determine if these species are protonated Fe(IV)oxos. These measurements will provide insight into Nature's use of thiolate-ligation in its hydroxylating heme enzymes.